Stem cells have different differentiation potency depending on origins of each stem cell, unlike general somatic cells. Among them, an embryonic stem cell is formed from a blastocyst which is one of the earliest stage of embryonic development, and the most important characteristic of the embryonic stem cell is pluripotent. That is, the embryonic stem cell may be differentiated into most types of cells present in the body including three germ layers of endoderm, mesoderm and ectoderm and regenerated.
However, the main weakness in the embryonic stem cell is that a technology of differentiation into a desired cell is not sufficient, and there is a possibility of forming cancer at the time of transplantation. Due to the problem above, there is a limitation in the development of a cellular therapeutic agent. Nevertheless, research into a technology of developing a therapeutic agent utilizing embryonic stem cells in response to shortage of transplant organs or treatment of genetic diseases and nonrenewable diseases has been continuously conducted.
Yamanaka group established a reprogramming pluripotent stem cell having similar properties to an embryonic stem cell from a fibroblast of a tail of a mouse by over-expressing four reprogramming transcription factors (Yamanaka, S, Cell, 126:663-76, 2006). Next year, the Yamanaka group and James A. Thomson group reported that a reprogramming pluripotent stem cell similar to the embryonic stem cell may be formed from a human somatic cell. The two groups used different transcription factors for inducing reprogramming, wherein the Yamanaka group used Oct-4/Sox-2/c-Myc/Klf-4 and Thomson group used Oct-4/Nanog/Sox-2/Lin28 (Cell. 131:861-72, 2007; Science., 318(5858):1917-20, 2007). In the early stage, viruses were utilized for over-expression in the reprogramming transcription factor. However, when developing a therapeutic agent using the virus, a stability problem occurred. After that, together with efforts for reducing the number of reprogramming factors, researches for development of a gene combination capable of increasing reprogramming efficiency and methods of inducing reprogramming without using viruses have been conducted.
However, a gene has various problems in that it is not easy to transfer the gene into a cell, in particular, it is significantly difficult to be permeated into a nucleus, a duration in which protein is expressed in a cell is short, and it is significantly difficult to artificially adjust the amount of protein to be expressed in a target cell (Verma, I. M. and Somia, N., Nature 389:239-242, 1997). In addition, as an example of methods of transferring the gene, there is an introgression method into a cell by an electrode disturbance by applying an electrical stimulation to a cell membrane. However, the introgression method has a disadvantage in that the number of cells which are stuck to each other due to the damage of the cell membrane and do not grow again corresponds to about 70%. The most commonly used method is a method in which a molecule having a gene surrounded with a lipid membrane as a liposome form is introduced into a cell by endocytosis. In this method, cytotoxicity is not largely caused as compared to an electrical stimulation method. However, when considering that permeation efficiency is significantly deteriorated, it is considered that this experimental method is not the best method, either. On the contrary to the cell permeable method, there is a method of transferring a gene by a virus. Treatment into a cell line which is a host, with Lenti-, Sendai-, Retro viruses as a carrier, allows easy transfer into a cell like a mechanism causing an infection into a human, but also allows genes of the viruses to be inserted into a chromosome. This method is also generally used when inducing over-expressing materials having a large molecular weight such as a therapeutic agent or a gene in a biological experiment in a cell. However, since this method is an experimental method based on viruses, an introgression efficiency is significantly excellent; meanwhile, at the time of being applied to a clinical testing, there is a limitation in that safety may not be secured. Therefore, a method of safer and effectively delivering materials having biological activity into a cytoplasm or a nucleus of a living cell has been demanded.
As a result of research for the demand, a cell permeable peptide (CPP) was suggested. Among researches, a transactivator of transcription (TAT) protein which is a kind of a human immunodeficiency virus type-1 protein has been substantially studied and it is known that TAT protein effectively passes through a cell membrane and is easily transferred into the cytoplasm. It is known that this function is provided due to property of a protein transduction domain, which is the middle domain of the TAT protein (Green, M. and Loewenstein, P. M., Cell, 55:1179, 1988; Ma, M. and Nath, A., J. Virol., 71:2495, 1997). However, it is believed that the TAT domain functions directly on the lipid double-layer of the cell membrane (Vines, E. et al., J. Biol. Chem. 272:16010, 1997). However, the CPP is a protein derived from the virus, which is concerned about toxicity. On the other hand, even in the cases of a peptide having amino acid sequences from 339th to 355th of an antennapedia (ANTP) protein derived from a vinegar fly (drosophila sp.) (Schwarze, S. R. et al., Trends. Pharmacol. Sci., 21:45, 2000) and an artificial peptide in which electrically positive amino acids are listed, cell permeability thereof was confirmed by experiments (Laus, R. et al., Nature Biotechnol., 18:1269, 2000).
However, after it was found that in the case of connecting the existing CPP with other peptides or proteins, transportation to the cell of the fusion protein is effective, various applications using the CPP were attempted (Korean Granted Patent No. 568,457), but there has been no attempt of preparing a reprogramming fusion protein in which the virus-derived TAT peptide is not used as a transporter in the country.
Therefore, the present inventors made an effort to develop a more effective method of preparing a reprogramming induced pluripotent stem cell, and found that in the case of directly delivering a cell permeable fusion protein to a target somatic cell instead of inducing the reprogramming of the target somatic cell through the delivery of the genes, the reprogramming of the somatic cell could be induced without genetic modification, such that an induced pluripotent stem cell in which stability of the gene is maintained was capable of being prepared, thereby completing the present invention.